Translationally Controlled Tumor Protein Interacts with the Third Cytoplasmic Domain of Na,K-ATPase α Subunit and Inhibits the Pump Activity in HeLa Cells

Abstract

Translationally controlled tumor protein (TCTP) is a growth-related protein under transcriptional as well as translational control. We screened a rat skeletal muscle cDNA library using yeast two-hybrid system and found that TCTP interacts with the third large cytoplasmic domain of ␣1 as well as ␣2 isoforms of Na,K-ATPase, believed involved in the regulation of Na,K-ATPase activity. Interaction between TCTP and Na,K-ATPase was confirmed by coimmunoprecipitation in yeast and mammalian cells. We also showed, using 86 Rb ؉ uptake assay, that overexpression of TCTP inhibited Na,K-ATPase activity in HeLa cells. Northern and Western blotting studies of HeLa cells transiently transfected with GFPtagged TCTP showed that overexpression of TCTP did not change mRNA and protein levels of Na,K-ATPase. Recombinant TCTP protein purified from an Escherichia coli expression system inhibited purified HeLa cell plasma membrane Na,K-ATPase in a dose-dependent manner. Using deletion analysis, we also found that the C-terminal 102-172-amino-acid region of rat TCTP that contains the TCTP homology region 2 is essential for its association with, and inhibition of, Na,K-ATPase. Na,K-ATPase, a multimembrane-spanning enzyme, is essential for maintaining transmembrane gradients of Na ϩ and K ϩ ions and thus for cell homeostasis (1). These ionic gradients serve to control essential cellular processes such as cell volume, membrane potential, and nutrient transport (2). In addition, Na,K-ATPase is involved in cell proliferation and differentiation, heart and vascular muscle contraction, and neurotransmitter and hormone secretion (3). Thus dysfunction of this enzyme can profoundly affect cell function. Na,K-ATPase is composed of a catalytic 110-kDa ␣ subunit and a glycosylated 40 -60-kDa ␤ subunit. The ␣ subunit contains binding sites for cations, ATP, and cardiac glycosides. It has been suggested that there might exist a diffusible cytoplasmic regulator of Na,K-ATPase activity, possibly modulated by protein kinases and hormones (4, 5). The third large cytoplasmic domain (CD3) 1 of Na,K-ATPase was proposed to be one of the domains involved in the regulation of its activity by insulin, thereby playing an important role in the catalytic function and regulation of this enzyme (6). Interactions between the Nterminal region of the Na,K-ATPase ␣ subunit with phosphoinositide-3 kinase (7), cytoplasmic domain 2 (CD2) and CD3 with ankyrin (8, 9), CD3 with cofilin (10), and purified Na,KATPase with actin (11) and adducin (12) have also been demonstrated.We looked for other cytoplasmic agents that might interact with the CD3 of Na,K-ATPase ␣ subunit and regulate its activity and found that translationally controlled tumor protein (TCTP) acts as a cytoplasmic repressor of Na,K-ATPase. TCTP is a growth-related protein, under tight transcriptional as well as translational control (13,14). It occurs as a 23-kDa protein in humans and has a 21-kDa homologue in mice but shows no significant homology with any other family of proteins. Based on structural studies o...

The Translational Controlled Tumour Protein TCTP: Biological Functions and Regulation

“…They reported that overexpression of TCTP in HeLa cells resulted in tyrosine phosphorylation of the epidermal growth factor receptor and in activation of both the Ras/Raf/ ERK and the PI3K/Akt pathways (Kim et al 2009b). In a more recent paper, they provided a link to the activity of TCTP in binding to and inhibiting the Na,KATPase, which they had observed earlier (Jung et al 2004). The authors found that TCTP in binding to the third cytoplasmic domain of the Na,K-ATPase results in release and activation of the protein kinase Src and consequently in the activation of the PI3K/Akt and the Ras/Raf/ERK pathways, as well as of additional signalling pathways .…”

“…Na-K-ATPase C-terminus Na-K-ATPase inhibition Jung et al (2004), Kim et al (2008a) Sorting nexin 6 (SNX6) Na-K-ATPase activation Yoon et al (2006) Ca 2+ N-and C-terminus Ca 2+ -scavenging Feng et al (2007a), Graidist et al (2007), Kim et al (2000) Vitamin-D3 1. Cooperation with other anti-apoptotic proteins.…”

“…Another important player in maintaining intracellular ion homeostasis that is influenced by TCTP is the membrane-bound Na + ,K + -ATPase. Kyunglim Lee's group discovered that TCTP interacts with the third cytoplasmic domain of the alpha subunit of the Na + ,K + -ATPase in HeLa cells and inhibits its activity (Jung et al 2004). They also identified another protein, sorting nexin 6 (SNX6), that binds to TCTP and neutralises TCTP's negative effector activity on the Na + ,K + -ATPase, but does not have an inhibitory activity towards the enzyme on its own (Yoon et al 2006).…”

Translationally controlled tumor protein induces human breast epithelial cell transformation through the activation of Src

“…We previously reported that translationally controlled tumor protein (TCTP) binding to the cytoplasmic domain of Na, K-ATPase a subunit results in the activation of EGFR and its downstream signaling pathways related to cell survival and motility (Jung et al, 2004;Kim et al, 2009). Thus, it appears that Na,K-ATPase inhibition by TCTP induces Src activation.…”

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

“…Several additional molecular interactions and regulatory functions have been reported for TCTP: (1) the protein binds to the Na þ , K þ -ATPase and inhibits this enzyme (Jung et al, 2004). This observation is consistent with the demonstration that TCTP overexpression in mice results in systemic hypertension (Kim et al, 2008b); (2) TCTP interacts with protein synthesis elongation factor eEF1A and negatively regulates the guanosine-nucleotide-exchange reaction on eEF1A (Cans et al, 2003); (3) a study using gene knockdown in Drosophila reported the activity of TCTP as a guanine-nucleotide-exchange factor for the small GTPase, Rheb, and indicated involvement of TCTP in the target of rapamycin (TOR) signalling pathway (Hsu et al, 2007); however, recent results from our own (Wang et al, 2008) and two other laboratories (Chen et al, 2007;Rehmann et al, 2008) are not compatible with this conclusion.…”